Cardiopulmonary Bypass Induced Inflammatory Changes in the Atrial Wall: The Novel Role for Cardiac Chymase produced Angiotensin II in the Development of Atrial Fibrillation

Michael Manning, MD, PhD

Over the last four months, I have been working to establish many of the tools that we will need to conduct these new and exciting experiments. First, we now have working protocols in place that will allow us to determine how the protein levels in the heart change during cardiopulmonary bypass, which we believe will result in structural changes in the heart leading to the development of post-operative atrial fibrillation.

In addition, we have similar protocols in place to evaluate how the specific genes in the renin-angiotensin-aldosterone system change during bypass. Both of these protocols were quite time consuming to develop, but now that we have them, we have used them to look at some very early samples taken from an animal model of cardiopulmonary bypass. So far, the results look very promising.

Next, we have demonstrated that we can reliably produce atrial fibrillation in a rat and trace it using an EKG printout. This is very exciting for us because we could be the first group to have an animal model of post-operative atrial fibrillation.

We have also been building collaborations with key individuals, both leaders in their fields, at Duke and Wake Forest. Dr. Dawn Bowles, Assistant Professor of Surgery at Duke, will be helping us with the acquisition of human atrial tissue samples from volunteers undergoing cardiac surgery with cardiopulmonary bypass. We will be using these tissue samples to directly correlate our findings from animal studies to humans. Dr. Mark Chappell, Professor of Surgical Sciences at Wake Forest, will be assisting us by measuring tissue levels of Angiotensin II with the hearts of both our animal and human tissues.

Balanced salt solutions are often used for the treatment of dehydration among patients undergoing surgical procedures, while intravenous saline solutions are the most commonly used fluid among patients with serious infections.

Our preliminary analysis of over 50,000 patients with serious infections admitted to intensive care units in over 300 hospitals across the United States between 2005-2010 shows that the use of balanced salt solutions is safer than the use of intravenous saline. In addition, there is a ‘dose-response’ relationship, such that as the proportion of balanced salt solutions increases instead of saline, the risk of death decreases. For instance, if patients receive five liters of fluid over the first day in the hospital, those receiving all five liters as balanced salt solutions do better than those receiving four liters of balanced salt solution with one liter of saline and so on. In this scenario, patients that receive five liters of saline have the worst outcomes.

The overall goal of this study was to understand how hibernating animals have developed natural defense mechanisms to withstand extremes of environment, and to ultimately apply this knowledge for organ protection in humans undergoing heart surgery.

We have previously characterized specific metabolic and anti-inflammatory adaptive responses to surgery in hibernating (arctic ground squirrels) versus non-hibernating animals (rats). Since the previous quarterly report, our team has conducted key additional experiments in summer active arctic ground squirrels to demonstrate that the organ protective mechanisms observed are a consequence of the hibernation state. We have received funding from the Foundation of Anesthesia Education and Research, and two additional grant applications are under review (an American Heart Association National Established Investigator grant, and an NIH Transformative-R01 grant). Our results have been selected for podium presentations at various international, national and local meetings, including the American Society of Anesthesiology and the American Heart Association.

In the upcoming quarter we plan to conduct additional experiments in both winter hibernating and summer active animals to characterize changes in the abundance of the full spectrum of proteins expressed in the heart using proteomic analyses, as well as in the repertoire of inflammatory cells responsible for heart damage following cardiac surgery using flow cytometry analyses.

Determinants of Intestinal Epithelial Wound Healing

Joern A. Karhausen, MD

The main hypothesis of our proposal was that the local interplay of hypoxic and inflammatory mechanisms in intestinal ischemia/reperfusion injury plays an important role in epithelial recovery through the action of the transcription factor ZEB-1.

Based on our extensive literature review, we had postulated that ZEB-1 activity is regulated in a feedback mechanism between ZEB-1 itself and a group of regulatory nucleic acids called microRNAs. However, extensive investigations in cell culture models, as well as in samples from mouse and rat models, were unable to support this initial view. Instead, our results suggest that ZEB-1 is stabilized through protein modifications, a mechanism that allows very rapid changes in a target protein stability or activity. To investigate these findings further, we have initiated a collaboration with Prof. Wulf Paschen, an expert in one of these pathways. His work had shown that in ischemia/reperfusion a group of small proteins called small ubiquitin like modifiers (SUMO) can dramatically alter cellular pathways. Through interacting with various targets these SUMO proteins can modify important cell-stress responses.

A better understanding of these mechanisms is a key to developing conceptually new therapeutic approaches that brace the intestinal barrier against ischemia/reperfusion injury, and we are currently seeking extra-mural funding to continue this novel and important work.

Effect of a Mn Porphyrin in Neuropathic Pain

With the support of our DIG, we developed two mouse models of spinal cord injury (SCI)-induced neuropathic pain in the lab.

The first model was related to spinal cord ischemia. Mice were subjected to aorta cross-clamping surgery and observed for two months. We observed that ischemic mice showed less tolerance to mechanical stimulus and were significantly different from non-injured mice. The preliminary data was used in a DOD grant proposal submitted in October, entitled, “Use of A Catalytic Oxidoreductant to Treat Neuropathic Pain after SCI.”

The second model was related to spinal cord trauma. The lumbar spinal cord in mice was damaged, and injured mice received either vehicle or Mn porphyrin daily treatment for four weeks. Behavioral tests were performed weekly. Our plan is to use the data from this study to write a new NIH grant and further study the effect of Mn porphyrin in attenuating neuropathic pain.